The present invention is related to the fields of molecular biology and oncology and provides methods for diagnosis, staging and monitoring of melanoma patients.
Cancer cells almost invariably undergo a loss of genetic material (DNA) when compared to normal cells. This deletion of genetic material which almost all, if not all, varieties of cancer undergo is referred to as xe2x80x9closs of heterozygosityxe2x80x9d (LOH). The loss of genetic material from cancer cells can result in the selective loss of one of two or more alleles of a gene vital for cell viability or cell growth at a particular locus on the chromosome. All genes, except those of the two sex chromosomes, exist in duplicate in human cells, with one copy of each gene (allele) found at the same place (locus) on each of the paired chromosomes. Each chromosome pair thus contains two alleles for any gene, one from each parent. This redundancy of allelic gene pairs on duplicate chromosomes provides a safety system; if a single allele of any pair is defective or absent, the surviving allele will continue to produce the coded protein.
Due to the genetic heterogeneity or DNA polymorphism, many of the paired alleles of genes differ from one another. When the two alleles are identical, the individual is said to be homozygous for that pair of alleles at that particular locus. Alternatively, when the two alleles are different, the individual is heterozygous at that locus. Typically both alleles are transcribed and ultimately translated into either identical proteins in the homozygous case or different proteins in the heterozygous case. If one of a pair of heterozygous alleles is lost due to a deletion of DNA from one of the paired chromosomes, only the remaining allele will be expressed and the affected cells will be functionally homozygous. This situation is termed a xe2x80x9closs of heterozygosityxe2x80x9d (LOH) or reduction to homozygosity. Following this loss of an allele from a heterozygous cell, the protein or gene product thereafter expressed will be homogeneous because all of the protein will be encoded by the single remaining allele. The cell becomes effectively homozygous at the gene locus where the deletion occurred. Almost all, if not all, cancer cells undergo LOH at some chromosomal regions.
Through the use of DNA probes, DNA from an individual""s normal cells can be compared with DNA extracted from the same individual""s tumor cells and LOH can be identified using experimental techniques well known in the art. Alternatively, LOH can be assayed by demonstrating two polymorphic forms of a protein in normal heterozygous cells, and only one form in cancer cells where the deletion of an allele has occurred. See for example Lasko, 1991.
Recent advances in molecular biology have revealed that genesis and progression of tumors follow an accumulation of multiple genetic alterations, including inactivation of tumor suppresser genes and/or activation of proto-oncogenes. There are over 40 known proto-oncogenes and suppressor genes to date, which fall into various categories depending on their functional characteristics. These include, growth factors and growth factor receptors, messengers of intracellular signal transduction pathways, for example, between the cytoplasm and the nucleus, and regulatory proteins influencing gene expression and DNA replication. Frequent loss of heterozygosity (LOH) on specific chromosomal regions has been reported in many kinds of malignancies, which indicates the existence of putative tumor suppresser genes or tumor-related genes on or near these loci. LOH analysis is a powerful tool to search for a tumor suppresser gene by narrowing and identifying the region where a putative gene exists. By now, numerous LOH analyses, combined with genetic linkage analysis on pedigrees of familial cancer (Vogelstein, 1988; Fearon, 1990; Friend, 1986) or homozygous deletion analyses (Call, 1990; Kinzler, 1991; Baker, 1989) have identified many kinds of candidate tumor suppresser or tumor-related genes. Also, because allelic losses on specific chromosomal regions are the most common genetic alterations observed in a variety of malignancies, microsatellite analysis has been applied to detect DNA of cancer cells in specimens from body fluids, such as sputum for lung cancer and urine for bladder cancer. (Rouleau, 1993; Latif, 1993) Moreover, it has been established that markedly increased concentrations of soluble DNA are present in plasma of individuals with cancer and some other diseases, indicating that cell free serum or plasma can be used for detecting cancer DNA with microsatellite abnormalities. (Kamp, 1994; Steck, 1997) Two groups have reported microsatellite alterations in plasma or serum of a limited number of patients with small cell lung cancer or head and neck cancer. (Hahn, 1996; Miozzo, 1996)
Recent developments in cancer therapeutics have demonstrated the need for more sensitive staging and monitoring procedures to ensure initiation of appropriate treatment, to define the end points of therapy and to develop and evaluate novel treatment modalities and strategies. In the management of melanoma patients, the choice of appropriate initial treatment depends on accurate assessment of the stage of the disease. Patients with limited or regional disease generally have a better prognosis and are treated differently than patients who have distant metastases (Minna, 1989). However, conventional techniques to detect these metastases are not very sensitive, and these patients are often not cured by primary tumor resection because they have metastases that are not identified by standard methods during preoperative staging. Thus, more sensitive methods to detect metastases in other types of carcinomas would identify patients who will not be cured by local therapeutic measures, for whom effective systemic therapies would be more appropriate.
The strategy of the present invention is to utilize genetic differences between normal and cancer cells for diagnosis and monitoring of melanoma patients. Many genes coding for proteins or other factors vital to cell survival and growth that are lost, can be identified through LOH analysis of microsatellite loci in cancer cells and mapped to specific chromosomal regions. In melanoma, mutations of several already-known tumor suppresser genes such as p53 gene, neurofibromatosis 1 (NF1) gene, and NF2 gene have been reported at a low frequency and deletions and/or mutations of the cyclin dependent kinase 4 (CDK4) inhibitor gene, which is a responsible tumor suppresser gene for a familial melanoma, have been thought to be important genetic changes in tumor development. (Miozzo, 1996) In addition to the locus of CDK4 inhibitor gene (9p21), frequent chromosomal deletions have been reported on 1p36, 3p25, 6q22-q26, 10q24-q26, and 11q23. (Mao, 1996; Stroun, 1987; Chen 1996; Nawroz, 1996)
Thus, an efficient method of testing DNA microsatellite loci for LOH allows early diagnosis of melanoma patients and monitoring of the progression of the disease as well as effectiveness of the therapeutic regimen.
It is an object of the invention to provide methods for identifying and assessing the extent of genetic change in neoplastic tissue. More specifically, the present invention provides methods for early diagnosis, staging and monitoring tumor progression and tumor genetic instability of melanoma patients by detecting the loss of a specified set of polymorphic alleles (LOH), alone or in combinations, in DNA from plasma and serum. In a preferred embodiment, this method comprises the steps of (a) in a sample of biological fluid, amplifying nucleic acid from an LOH marker, if present, (b) detecting the presence or absence of the LOH marker, and (c) correlating the findings with the occurrence and/or progression of melanoma. In a preferred embodiment of the present invention, the set of alleles which are tested for LOH are selected from the group consisting of D1S214, D1S228, D3S1293, D6S264, D9S157, D9S161, S10S212, D10S216, D11S925. In addition, combinations of the alleles, including D9S157 combined with D3S1293, D9S157 combined with D1S228, D1S925 combined with D3S1293, and all alleles are tested.
It is another object of the invention to provide a kit for diagnosing, staging and monitoring melanoma patients. In a preferred embodiment of the invention, a kit is provided comprising a set of nucleic acid probes for specified alleles for which the patient is homozygous or heterozygous to detect LOH in these specified alleles. This will provide a measure of the extent of genetic change in the neoplastic tissue which can be correlated with a prognosis. In one specific embodiment, the presence or absence of a specific allele or combination of alleles is tested by amplification of regions of the DNA using pairs of primers which bracket specific regions of DNA on specific chromosome arms containing repeat sequences with polymorphism. Preferably the assay uses fluorescent labeling of DNA with multiple types of chromophores. However, radioactive and other labeling techniques known in the art also may be used.
This invention provides a logistically practical assay to monitor the genetic changes during melanoma progression. The events of tumor progression are dynamic and the genetic changes that concurrently occur also are very dynamic and complex. The most significant advantage of this approach compared to other approaches in the ability to monitor disease progression and genetic changes without assessing the tumor. This is particularly important during early phases of distant disease spread, in which subclinical disease is undetectable by conventional imaging techniques. In addition, in advance stage diseases or inoperable sites in which tumor tissue is very difficult or impossible to obtain for genetic analysis, the present invention provides an alternative for assessing LOH.